蛋白激酶C-α在调节人膀胱癌T24细胞多药耐药中的作用

The role of protein kinase C- alpha in regulating multidrug resistance in human bladder cancer cell line T24

目的探讨蛋白激酶C-α(PKCα)对人膀胱癌T24细胞多药耐药的调节作用。方法将载有PKCα基因的表达质粒GFP-PKCα导入人膀胱癌细胞系T24。应用噻唑蓝(MTT)比色法,检测T24/PKCα及亲本细胞对阿霉素的敏感性,以及在激活(10nmol/LPMA2h)和抑制(10nmol/LCalphostinC2h)PKCα的情况下,T24/PKCα细胞对阿霉素敏感性的变化。同时用逆转录-聚合酶链反应(RT-PCR)检测多药耐药基因-1(MDR-1)、多药耐药相关蛋白(MRP)和肺耐药相关蛋白(LRP)基因的表达。结果转染了PKCα基因后,T24细胞对阿霉素的耐药性增强(耐药指数7.52,P<0.05)。PKCα的激活显著提高了T24细胞的耐药性(耐药指数153.78,P<0.01),而抑制PKCα活性则使耐药指数降至1.20(P>0.05)。T24/PKCα的MDR-1表达水平是亲本细胞的2.28倍(P<0.01)。激活PKCα可使MDR-1基因表达水平较亲本细胞升高12.80倍(P<0.01),抑制PKCα则MDR-1表达下降(P>0.05),而MRP和LRP基因表达水平并无明显变化(P>0.05)。结论PKCα可能通过上调MDR-1表达,在人膀胱癌的化疗耐药中起到了重要的作用。

Objective To investigate the role of protein kinase C- alpha (PKCα) in regulating multidrug resistance (MDR) in human bladder cancer cell line T24.Methods Plasmid green fluorescent protein -PKCα (GFP/PKCα) carrying PKCα gene was transfected into a human T24 bladder cancer cell line.The sensitivity of the T24/PKCα and parental cells to adriamycin was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.The change of sensitivity of the T24/PKCα to adriamycin under the conditions of PKCα activation (10 nmol/L PMA 2 h) and inhibition (10 nmol/L Calphostin C 2 h) was also observed respectively.Meanwhile reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of multidrug-resistance gene1 (MDR-1),multidrug resistance-associated protein (MRP) and lung resistance-related protein (LRP).Results Transfection of PKCα increased resistance of T24 cells to adriamycin with the resistance index (RI) being 7.52 (P< 0.05). PKCα activation further greatly promoted the resistance with the RI being 153.78 (P< 0.001), while inhibition of PKCα did conversely with the RI being 1.2 (P> 0.05). The expression of MDR-1 was 2.28-fold higher in T24/PKCα than in its parent cell line (P< 0.01). PKCα activation further greatly increased 12.28-fold MDR-1 expression in T24/PKCα than in its parent cell line (P< 0.01), while inhibition of PKCα did conversely (P> 0.05). There was no significant difference in the expression of MRP and LRP between the cell lines (P> 0.05). Conclusion PKCα might play an important role in resistance to chemotherapy drugs in human bladder carcinoma via up-regulation of the MDR-1 expression.